The present invention relates to:
I. a novel transferase, a process for producing the same, a process for producing an oligosaccharide by using the enzyme, a gene coding for the enzyme, and use thereof; and
II. a novel amylase, a process for producing the same, a process for producing xcex1,xcex1-trehalose by using the enzyme, a gene coding for the enzyme, and use thereof. More specifically, as follows.
I. The present invention relates to a novel transferase which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to transfer the xcex1-1,4 linkages to xcex11,xcex1-1 linkages; and a process for producing the transferase. More particularly, the present invention relates to the above-mentioned enzyme produced from archaebacteria belonging to the order Sulfolobales, for example, bacteria of the genus Sulfolobus or Acidianus.
Further, the present invention relates to a novel process for producing trehaloseoligosaccharides or the like by using the above-mentioned novel enzyme, and more particularly, relates to an efficient and high-yield process for producing trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehaloses by using a maltooligosaccharide or the like as a raw material.
Moreover, the present invention relates to a DNA fragment coding for the above-mentioned novel transferase and to the use of the DNA fragment in genetic engineering.
II. The present invention relates to a novel amylase which acts on a substrate saccharide, the saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end are glucose residues, so as to liberate principally monosaccharides and/or disaccharides by hydrolyzing the substrate from the reducing end; and a process for producing the amylase. More particularly, the present invention relates to a novel amylase which has an principal activity of acting on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end side are glucose residues and the linkage between the first and the second glucose residues from the reducing end side is xcex1-1,xcex1-1 while the linkage between the second and the third glucose residues from the reducing end side is xcex1-1,4, so as to liberate xcex1,xcex1-trehalose by hydrolyzing the xcex1-1,4 linkage between the second and the third glucose residues; and a process for producing the amylase. The novel amylase also has another activity of endotype-hydrolyzing one or more xcex1-1,4 linkages within the molecular chain of the substrate, and can be produced by bacteria belonging to the genus Sulfolobus. This enzyme is available for the starch sugar industry, textile industry, food industry, and the like.
Further, the present invention relates to a process for producing xcex1,xcex1-trehalose, characterized by using the above novel amylase in combination with the above novel transferase. In detail, the present invention relates to a process for producing xcex1,xcex1-trehalose in a high yield by using, as a raw material, any one of starch, starch hydrolysate and maltooligosaccharides, or a mixture of maltooligosaccharides, and as enzymes, the novel transferase and amylase of the present invention.
Moreover, the present invention relates to a DNA fragment coding for the above novel amylase, and use of the DNA fragment in genetic engineering.
I. Background Art of Transferase
Hitherto, in relation to glycosyltransferase acting on starch and starch hydrolysates such as maltooligosaccharides, various glucosyltransferases, cyclodextringlucanotransferases (CGTase), and others have been found [c.f. xe2x80x9cSeibutsu-kagaku Jikken-houxe2x80x9d 25 (xe2x80x9cExperimental Methods in Biochemistryxe2x80x9d, Vol. 25), xe2x80x98Denpun.Kanren Toushitsu Kouso Jikken-houxe2x80x99 (xe2x80x98Experimental Methods in Enzymes for Starch and Relating Saccharidesxe2x80x99), published by Gakkai-shuppan-sentah, Bioindustry, Vol. 9, No. 1 (1992), p. 39-44, and others]. These enzymes transfer a glucosyl group to the xcex1-1,2, xcex1-1,3, xcex1-1,4, or xcex1-1,6 linkage. However, an enzyme which transfers a glucosyl group to the xcex1-1,xcex1-1 linkage has not been found yet. Though trehalase has been found as an enzyme which acts on the xcex1-1,xcex1-1 linkage, trehalose is absolutely the only substrate for the enzyme, and the equilibrium or the reaction rate lies to the degrading reaction.
Recently, oligosaccharides were found to have physicochemical properties such as moisture-retaining ability, shape-retaining ability, viscous ability and browning-preventive ability, and bioactivities such as a low-calorigenetic property, an anticariogenic property and a bifidus-proliferation activity. In relation to that, various oligosaccharides such as maltooligosaccharides, branched-chain oligosaccharides, fructooligosaccharide, galacto-oligosaccharide, and xylooligosaccharide have been developed [c.f. xe2x80x9cKammiryoxe2x80x9d (xe2x80x9cSweetenerxe2x80x9d) (1989), Medikaru-risahchi-sha (Medical Research Co.) (1989), Gekkan Fuhdokemikaru (Monthly Foodchemical) (1993), February p. 21-29, and others].
Among oligosaccharides, the oligosaccharides which have no reducing end may include fructooligosaccharide having a structure composed of sucrose which is not reductive, and being produced by fructosyltransferase. Meanwhile, among starch hydrolysates such as maltooligosaccharides, the oligosaccharides which have no reducing end may include cyclodextrins produced by the above-mentioned CGTase, xcex1,xcex2-trehalose (neotrehalose), and reduced oligosaccharides chemically synthesized by hydrogenating the reducing end (oligosaccharide alcohol). These oligosaccharides having no reducing end have various physicochemical properties and bioactivities which are not possessed by conventional starch syrups and maltooligosac-charides. Accordingly, among maltooligosaccharides, the oligosaccharides the reducing ends of which are modified with an xcex1-1,xcex1-1 linkage may be also expected to have the similar physicochemical properties and bioactivities to those possessed by the above-mentioned oligosaccharide having no reducing end, since such oligosaccharides also have no reducing end.
Here, the oligosaccharides the reducing ends of which are modified with an xcex1-1,xcex1-1 linkage as described above may be recognized as a trehaloseoligosaccharide in which xcex1,xcex1-trehalose is linked with glucose or a maltooligoshaccharide. Accordingly, such a trehaloseoligosaccharide may be expected to have the physicochemical properties and bioactivities which are possessed by the oligosaccharide having no reducing end, and in addition, may be expected to have the specific activities as exhibited by xcex1,xcex1-trehalose (c.f. Japanese Patent Laid-open Publication No. 63-500562).
Though it was reported that a trace amount of trehaloseoligosaccharides could be detected in yeast [Biosci. Biotech. Biochem., 57(7), p. 1220-1221 (1993)], this is the only report referring to its existence in nature. On the other hand, as to its synthesis by using an enzyme, though there has been a report of such synthesis [Abstracts of xe2x80x9c1994 Nihon Nougei-kagaku Taikaixe2x80x9d (xe2x80x9cAnnual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry in 1994xe2x80x9d), p. 247], the method described in the report uses trehalose, which is expensive, as the raw material. Therefore, production at low cost has not yet been established.
Recently, Lama, et al. found that a cell extract from the Sulfolobus solfataricus strain MT-4 (DSM 5833), a species of archaebacteria, has a thermostable starch-hydrolyzing activity [Biotech. Forum. Eur. 8, 4, 2-1 (1991)]. They further reported that the activity is also of producing trehalose and glucose from starch. The above-mentioned report, however, does not at all refer to the existence of trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehalose. Moreover, no investigation in archaebacteria other than the above-mentioned strain has been attempted.
Meanwhile, an efficient process for obtaining the novel transferase should be established to efficiently produce trehaloseoligosaccharides.
Accordingly, mass-production of trehaloseoligosaccharides requires obtaining this novel transferase in a large amount. For achievement of this, it is preferable to obtain a gene coding for such transferase, and to produce the transferase in a genetic engineering manner. When such a gene can be obtained, it can be also expected, by using technologies of protein engineering, to obtain an enzyme having an improved thermostability, an improved pH stability, and an enhanced reaction rate. No report has, however, been made about gene cloning of such a gene yet.
An object of the present invention is to provide a novel transferase principally catalyzing the production of trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehaloses, and a process for producing the enzyme, and further, to provide a novel, efficient and high-yield process for producing principally trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehaloses by using such an enzyme from a raw material such as maltooligosaccharides.
Inventors earnestly investigated the trehalose-producing activity of archaebacteria and found that glucosyltrehalose can be produced from maltotriose as a substrate by cell extracts from various archaebacteria such as those belonging to the order Sulfolobales, and more specifically, the genera Sulfolobus, Acidianus, and others. Here, though production of trehalose and glucose was confirmed using an activity-measuring method described by Lama, et al. in which the substrate is starch, Inventors found that detection of trehaloseoligosaccha-rides such as glucosyltrehalose is extremely difficult. Also, Inventors found that the trehalose-producing activity as found by Lama, et al. disappears during the step for purification of cell extracts from archaebacteria. Consequently, the inventors recognized that the purification and characterization of the enzymes themselves which have such activities were substantially impossible.
Under such circumstances, Inventors made further investigations and conceived a novel activity-measuring method in which the substrate is a maltooligosaccharide such as maltotriose, and the index is activity of producing a trehaloseoligosaccharide such as glucosyl-trehalose. Then, it was found by a practice of the measuring method that a trehaloseoligosaccharide such as glucosyltrehalose can be easily detected. Further, the Inventor attempted to purify the enzyme having such activity from various bacterial strains, and found, surprisingly, that the enzyme thus obtained is quite a novel transferase which acts on maltotriose or a larger saccharide wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, and which transfers the linkage between the glucose residues at the reducing end into an xcex1-1,xcex1-1linkage to produce trehaloseoligosaccha-rides such as glucosyltrehalose. Incidentally, the existence of trehaloseoligosaccharides which are produced from maltooligosaccharides or the like by transferring the linkage between glucose residues at the reducing end into an xcex1-1,xcex1-1linkage was confirmed by 1H-NMR and 13C-NMR (c.f. Examples I-1, 7 and 8).
Inventors further found that such a novel enzyme is available for producing a large amount of trehaloseoligosaccharides, for example, glucosyltrehalose and maltooligosyltrehalose from saccharides such as maltooligosaccharides, and have accomplished the present invention.
Moreover, Inventors isolated the genes coding for such a novel enzyme, and have now established a process for producing the novel transferase by using such genes in a genetic engineering manner.
II. Background Art of Amylase
xe2x80x9cAmylasexe2x80x9d is a generic term for the enzymes which hydrolyze starch. Among them, xcex1-amylase is an enzyme which endotype-hydrolyzes an xcex1-1,4 glucoside linkage. Alpha-amylase widely exists in the living world. In mammals, xcex1-amylase can be found in saliva and pancreatic fluid. In plants, malt has the enzyme in large amounts. Further, xcex1-amylase widely exists in microorganisms. Among them, xcex1-amylase or the like which is produced by some fungi belonging to the genus Aspergillus or some bacteria belonging to the genus Bacillus is utilized in the industrial fields [xe2x80x9cAmirahzexe2x80x9d (xe2x80x9cAmylasexe2x80x9d), edited by Michinori Nakamura, published by Gakkai-shuppan-sentah, 1986].
Such xcex1-amylase is industrially and widely used for various purposes, for example, for starch-liquefying processes in starch sugar industries, and for desizing processes in textile industries, and therefore, the enzyme is very important from an industrial view. The following are listed as important conditions for the starch-liquefying process in xe2x80x9cKouso-Ouyou no Chishikixe2x80x9d (written by Toshiaki Komaki, published by Sachi-Shobou, 1986): 1) the starch molecules should be liquefied as completely as possible, 2) the products produced by the liquefaction are favorable for the purpose of the subsequent saccharifying process, 3) the condition does not cause retrogradation of the products by the liquefaction, and 4) the process should be carried out in a high concentration as much as possible (30-35%) in view of reducing cost. A starch-liquefying process may be performed, for example, by a continuous liquefaction method at a constant temperature, or by the Jet-Cooker method. Ordinarily, a thick starch-emulsion containing xcex1-amylase is instantaneously heated to a high temperature (85-110xc2x0 C.), and then the xcex1-amylase is put into action to perform liquefaction at the same time as starch begins to be gelatinized and swollen. In other words, the starch-liquefying process requires a temperature sufficient to cause the starch to swell before the enzyme can act. Enzymes capable of being used in such fields are, for example, the above-mentioned thermostable xcex1-amylases produced by fungi of the Aspergillus oryzae group belonging to the genus Aspergillus or bacteria belonging to the genus Bacillus. In some cases, the addition of calcium is required for further improving thermostability of these enzymes. In the starch-liquefying process, once the temperature declines while the xcex1-amylase has not yet acted on the starch-micelles which are swelled and going to be cleaved, starch will be agglutinated again to form new micelles (insoluble starch) which are rarely liquefied by xcex1-amylase. As a result, the liquid sugar thus produced will be turbid and hard to filtrate, as is a known problem. Some methods which increase the liquefaction degree, i.e. dextrose equivalent (DE), are used in order to prevent such an event. However, in some cases, such as an enzymatic production of maltose, DE should be maintained as low as possible, namely, the polymerization degree of the sugar chain should be maintained to a high degree in order to keep a high yield. Accordingly, when an enzyme is further used for a process subsequent to a starch-liquefying process, use of an enzyme thermostable enough for use in a series of high temperatures will allow the progress of the reaction without producing slightly soluble starch even by using a high concentration of starch, and at the same time, such use will be advantageous in view of process control and sanitary control because the risk of contamination with microorganisms can be decreased. Meanwhile, when the enzyme is immobilized in a bioreactor to use the enzyme recyclically, it is believed to be important that the enzyme has high stability, and especially high thermostability, since the enzyme may be exposed to a relatively high temperature during immobilization. If the enzyme has a low thermostability, it will possibly be inactivated during the immobilization procedure. As is obvious from the above, an enzyme having a high thermostability can be used very advantageously in several industrial fields, for example, a starch-liquefying process, and such an enzyme is desired.
In addition, screening of thermophilic and hyper-thermophilic bacteria has been widely carried out in recent years in order to obtain thermostable enzymes including amylase. Archaebacteria belonging to the order Thermococcales and the genus Pyrococcus are also the objects of screening, and were reported to produce xcex1-amylase [Applied and Environmental Microbiology, pp.1985-1991, (1990); Japanese Patent Laid-open Publication No. 6-62869; and others]. Additionally, archaebacteria belonging to the genus Sulfolobus are the objects of screening, and isolation of thermostable enzymes was reported. Here, archaebacteria belonging to the genus Sulfolobus are taxonomically defined by the following characteristics:
being highly thermophilic: being possible to grow in a temperature range of 55xc2x0 C.-88xc2x0 C.;
being acidophilic: being possible to grow in a pH range of 1-6;
being aerobic; and
being sulfur bacteria: being cocci having irregular form, and a diameter of 0.6-2 xcexcm. Accordingly, if an archaebacterium belonging to the genus Sulfolobus produces an amylase, the amylase is expected to be also thermo-stable. Lama, et al.found that a thermostable starch-hydrolyzing activity exists in a cell extract from the Sulfolobus solfataricus strain MT-4 (DSM 5833) [Biotech. Forum. Eur. 8, 4, 2-1 (1991)]. This article reported that xcex1,xcex1-trehalose and glucose can be produced from starch by this activity. However, purification of the active substance was performed only partially, and the true substance exhibiting the activity has not yet been identified. In addition, the enzymatic characteristics of the activity has not been clarified at all. The Inventors"" investigations, the details of which will be described below, revealed that the active substance derived from the above-mentioned bacterial strain and allowed to act on starch by Lama, et al. was a mixture containing a plurality of enzymes, and that xcex1,xcex1-trehalose and glucose are the final products obtained by using the mixture.
As another characteristic, xcex1-amylase has an activity of, at an initial stage, decreasing the quantity of iodo-starch reaction, namely, an activity of endotype-hydrolyzing xcex1-1,4-glucan (liquefying activity). There are several modes in the reaction mechanism of such liquefying-type amylase. In other words, it is known that each amylase has common characteristics in view of endotype-hydrolyzing activity but has individual characteristics in view of patterns for hydrolyzing maltooligosaccharides. For example, some recognize a specific site for hydrolysis of the substrate from the non-reducing end, and others recognize a specific site for hydrolysis of the substrate from the reducing end. Further, some hydrolyze the substrate to principally produce glucose, others to principally produce maltose or maltooligosaccharides. More specifically, the xcex1-amylase derived from pancreas hydrolyzes the xcex1-1,4 linkage second or third from the reducing end [xe2x80x9cDenpun.Kanren Toushitsu Kouso Jikken-houxe2x80x9d (xe2x80x9cExperimental methods in enzymes for starch and relating saccharidesxe2x80x9d), written by Michinori Nakamura and Keiji Kainuma, published by Gakkai-Shuppan-Sentah, 1989]. The xcex1-amylase derived from Bacillus subtilis hydrolyzes the xcex1-1,4 linkage sixth from the non-reducing end or third from the reducing end [xe2x80x9cKouso-Ouyou no Chishikixe2x80x9d (xe2x80x9cKnowledge in Application of Enzymesxe2x80x9d), written by Toshiaki Komaki, published by Sachi-Shobou, 1986]. It is believed that such a difference between the reaction modes of xcex1-amylases can be attributed to the structure of each enzyme, and the xe2x80x9cSubsite theoryxe2x80x9d is proposed for explanation of these events. Additionally, the existence of an xcex1-amylase having transferring activities or condensation activities has been confirmed. Further, a particular xcex1-amylase which produces a cyclodextrin has been found.
On the other hand, xcex1,xcex1-trehalose consists of two glucose molecules which are xcex1-1,xcex1-1-linked together at the reducing group of each molecule. It is known that xcex1,xcex1-trehalose exists in many living things, plants and microorganisms of the natural world, and has many function such as preventing the biomembrane from freezing or drying, and being an energy source in insects. Recently, xcex1,xcex1-trehalose was evaluated in the fields of medicine, cosmetics and food as a protein stabilizer against freezing and drying (Japanese Examined Patent Publication No. 5-81232, Japanese Patent Laid-open Publication No. 63-500562, and others). However, xcex1,xcex1-trehalose is not often used practically. This may be because no mass-productive process has been established yet.
Examples of the conventional process for producing xcex1,xcex1-trehalose are as follows:
a process comprising extraction from an yeast (Japanese Patent Laid-open Publications Nos. 5-91890 and 4-360692, and others);
a process comprising intracellular production by an yeast (Japanese Patent Laid-open Publication No. 5-292986, European Patent No. 0451896, and others); and
a process comprising production by a microorganism belonging to the genus Sclerotium or the genus Rhizoctonia (Japanese Patent Laid-open Publication No. 3-130084). However, these processes, as comprising intracellular production, require a purification process comprising multiple steps for spallation of bacterial bodies and removal of debris. Meanwhile, several investigations were made into extracellular production by a fermentation using a microorganism, for example, a microorganism belonging to the genus Arthrobacter (Suzuki T, et al., Agric. Biol. Chem., 33, No. 2, 190, 1969) or the genus Nocardia (Japanese Patent Laid-open Publication No. 50-154485), and glutamate-producing bacteria (French Patent No. 2671099, Japanese Patent Laid-open Publication No. 5-211882, and others). Further, production by a gene encoding an enzyme for xcex1,xcex1-trehalose metabolism was attempted (PCT Patent No. 93-17093). Any of the above processes use glucose or the like as the sugar source, and utilize a metabolic system which requires ATP and/or UTP as the energy source. These processes, therefore, require a complicated purification process to obtain xcex1,xcex1-trehalose from the culture medium. Moreover, some investigations were attempted into production by an enzymatic process using, for example, trehalose phosphorylase (Japanese Examined Patent Publication No. 63-60998), or trehalase (Japanese Patent Laid-open Publication No. 7-51063). These processes, however, have some problems in mass-production of the enzymes, stability of the enzymes, and others. All of the processes of the prior art as described above have problems such as a low yield, complexity in the purification process, low production, and complexity in preparation of the enzyme. Therefore, a process having industrial applicability has not been established yet. Under the circumstances, a process for more efficiently producing xcex1,xcex1-trehalose is strongly desired to be established.
As described above, xcex1,xcex1-trehalose was found widely in nature, and the existence of it in archaebacteria was also confirmed (System. Appl. Microbiol. 10, 215, 1988). Specifically, as mentioned above, Lama, et al. found that a thermostable starch-hydrolyzing activity exists in a cell extract from an archaebacterium species, the Sulfolobus solfataricus strain MT-4 (DSM 5833), and confirmed the existence of xcex1,xcex1-trehalose in the hydrolyzed product [Biotech. Forum. Eur. 8, 4, 2-1 (1991), cited before]. This article reported that the activity was of producing xcex1,xcex1-trehalose and glucose from starch. The article, however, actually reported only an example in which the substrate was 0.33% soluble starch, the amount of xcex1,xcex1-trehalose produced thereby was extremely small, and besides, the ratio of produced xcex1,xcex1-trehalose to produced glucose was 1:2. Accordingly, an isolation process is necessary to remove glucose which is produced in a large amount as a by-product, and the purpose of establishing a process for mass-producing xcex1,xcex1-trehalose cannot be achieved at all.
Inventors, as described above, found that an archaebacteria belonging to the order Sulfolobales produce a transferase which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to transfer the first xcex1-1,4 linkage from the reducing end into an xcex1-1,xcex1-1linkage. Further, Inventors invented a process for producing trehaloseoligosaccharides such as glucosyltrehalose and maltooligosyltrehaloses from maltooligosaccharides by using this enzyme. Here, the trehaloseoligosaccharide is a maltooligosaccharide the reducing end side of which is modified with an xcex1-1,xcex1-1 linkage.
In the meantime, no report has been made, as far as Inventors know, as to an formerly-known enzyme capable of acting on a trehaloseoligosaccharide which is derived from a maltooligosaccharide by transforming the first linkage from the reducing end into an xcex1-1,xcex1-1 linkage, and capable of hydrolyzing specifically the xcex1-1,4 linkage next to the xcex1-1,xcex1-1 linkage to liberate xcex1,xcex1-trehalose in a high yield. In other words, conventional amylase cannot hydrolyze trehaloseoligosaccharide specifically at the xcex1-1,4 linkage between the second and third glucose residues from the reducing end side to liberate xcex1,xcex1-trehalose. It will, therefore, markedly benefit the mass-production of xcex1,xcex1-trehalose if an amylase can be developed, such amylase being capable of catalyzing the reaction for producing xcex1,xcex1-trehalose as well as hydrolyzing the xcex1-1,4 linkage in the molecular chain of starch or starch hydrolysate.
In addition, mass-production of xcex1,xcex1-trehalose requires obtaining the novel amylase in a large amount. For this purpose, it is preferable to obtain a gene coding for the amylase and to produce the enzyme in a genetic engineering manner. Further, if such a gene can be obtained, it can also be expected to obtain, by using a technology of protein engineering, an enzyme which has improved thermostability, improved pH stability, and an enhanced reaction rate.
An object of the present invention is to provide a novel amylase which has an activity of endotype-hydrolyzing the xcex1-1,4 linkage in the molecular chain of starch or starch hydrolysate, and which can catalyze the reaction of liberating xcex1,xcex1-trehalose, wherein the enzyme acts on a trehaloseoligosaccharide which is derived from a maltooligosaccharide by transforming the first linkage from the reducing end into an xcex1-1,xcex1-1linkage, and hydrolyzes specifically the xcex1-1,4 linkage between the second and third glucose residues from the reducing end side, and is to provide a process for producing such an enzyme. Another object of the present invention is to provide a novel process for efficiently producing xcex1,xcex1-trehalose in a high yield from a low-cost raw material such as starch, starch hydrolysate, and maltooligosaccharides by using the enzyme.
Inventors energetically investigated starch-hydrolyzing activity derived from archaebacteria. As a result, Inventors found that a thermostable starch-hydrolyzing activity exists in cell extracts from various archaebacteria belonging to the order Sulfolobales, and more specifically, the genus Sulfolobus. The saccharides produced by hydrolysis of starch were found to be glucose and xcex1,xcex1-trehalose, similar to the description in the article by Lama, et al. Inventors then examined extracts from various bacterial strains for characteristics of the starch-hydrolyzing activity. As a result, Inventors found that the enzymes produced by those strains are mixtures of enzymes comprising various endotype or exotype amylases such as liquefying amylase and glucoamylase, and transferase, in view of enzymatic activity such as starch-hydrolyzing activity and xcex1,xcex1-trehalose-producing activity. In addition, such enzymatic activities were found to be attributed to synergism by activities of these mixed enzymes. Further, when the activity-measuring method proposed by Lama, et al. is employed in purification of each enzyme, in which the index is decrement of blue color derived from iodo-starch reaction, the purification of each enzyme having such an activity resulted in a low yield on the whole, and such purification procedure was found to be very difficult. These events may be attributed to low sensitivity and low quantifying ability of the activity-measuring method. Moreover, the Inventors"" strict examination revealed that purification and isolation could not be accomplished at all, in terms of protein, by the partial-purification method described in the article by Lama, et al.
Under such circumstances, Inventors have made further investigation, and conceived a new activity-measuring method in which the substrate is a trehaloseoligosaccharide such as maltotriosyltrehalose, and the index is activity of liberating xcex1,xcex1-trehalose. By a practice of this measuring method, it was revealed that amylase activity can be easily detected using such a method. Inventors then tried to achieve purification of the enzyme having such an activity in various bacterial strains, and finally, succeeded in purification and isolation of such an amylase. Further, Inventors examined enzymatic characteristics of the isolated and purified amylase, and found, surprisingly, that the enzyme thus obtained has a novel action mechanism, namely, has the following characteristics together:
The enzyme exhibits an activity of endotype-hydrolyzing starch or starch hydrolysate;
the enzyme exhibits an activity of hydrolyzing starch hydrolysate, a maltooligosaccharide or the like from the reducing end to produce monosaccharides and/or disaccharides;
the enzyme exhibits a higher reactivity to a saccharide which is composed of at least three sugar units wherein the linkage between the first and second glucose residues from the reducing end side is xcex1-1,xcex1-1, and the linkage between the second and third glucose residues from the same end side is xcex1-1,4 (for example, trehaloseoligosaccharides), as compared with the reactivity to each of the corresponding maltooligosaccharides; and
the enzyme has an activity of acting on such substrate saccharides composed of at least three sugar units so as to liberate xcex1,xcex1-trehalose by hydrolyzing the xcex1-1,4 linkage between the second and third glucose residues from the reducing end side.
Moreover, Inventors isolated a gene coding for such novel enzyme, and now, have established a process for producing, in a genetic engineering manner, a recombinant novel amylase by utilizing such a gene.
I. Novel Transferase
The present invention provides a novel transferase (hereinafter referred to as xe2x80x9cnovel transferase of the present inventionxe2x80x9d, or simply referred to as xe2x80x9cthe enzyme of the present inventionxe2x80x9d or xe2x80x9cthe present enzymexe2x80x9d) which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to transfer the first xcex1-1,4 linkage from the reducing end into an xcex1-1,xcex1-1 linkage.
In another aspect, the present invention provides a novel transferase which acts on a substrate maltooligosaccharide, all of the constituting glucose residues of the maltooligosaccharide being xcex1-1,4-linked, so as to transfer the first xcex1-1,4 linkage from the reducing end into an xcex1-1,xcex1-1linkage.
Further, the present invention provides a process for producing the novel transferase of the present invention, wherein a bacterium capable of producing a transferase having such activities is cultivated in a culture medium, and the transferase is isolated and purified from the culture on the basis of an activity-measuring method in which the substrate is a maltooligosaccharide, and the index is the activity of producing trehaloseoligosaccharides.
Moreover, the present invention provides a process for producing a saccharide having an end composed of a couple of xcex1-1,xcex1-1-linked sugar units, characterized in that the enzyme of the present invention is used and allowed to act on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to produce the objective saccharide in which at least three sugar units from the reducing end side are glucose residues and the linkage between the first and second glucose residues from the reducing end side is xcex1-1,xcex1-1 while the linkage between the second and third glucose residues from the reducing end side is xcex1-1,4.
Furthermore, the present invention provides a process for producing a trehaloseoligosaccharide, wherein the enzyme of the present invention is used, and the substrate is each of maltooligosaccharides or a mixture thereof.
Additionally, an object of the present invention is to provide a gene coding for the transferase.
Further, another object of the present invention is to provide a recombinant novel transferase and a process for producing the same by using the above-mentioned gene.
Moreover, an object of the present invention is to provide an efficient process for producing trehaloseoligosaccharides such as glucosyltrehalose and maltoglucosyltrehalose by using a recombinant novel transferase.
Accordingly, the DNA fragment based on the present invention comprises a gene coding for a novel transferase which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to transfer the first xcex1-1,4 linkage from the reducing end into an xcex1-1,xcex1-1 linkage.
Further, the recombinant novel transferase according to the present invention is the product achieved by expression of the above-mentioned DNA fragment.
Moreover, the process for producing a recombinant novel transferase according to the present invention comprises:
culturing a host cell transformed with the above-mentioned gene;
producing said recombinant novel transferase in the culture; and
collecting the products.
II. Novel Amylase
The present invention provides a novel amylase which acts on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end are glucose residues, so as to liberate principally monosaccharides and/or disaccharides by hydrolyzing the substrate from the reducing end side.
In another aspect, the present invention provides a novel amylase which has a principal activity of acting on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end side are glucose residues and the linkage between the first and the second glucose residues from the reducing end side is xcex1-1,xcex1-1 while the linkage between the second and the third glucose residues from the reducing end side is xcex1-1,4, so as to liberate xcex1,xcex1-trehalose by hydrolyzing the xcex1-1,4 linkage between the second and the third glucose residues.
Further, in another aspect, the present invention provides a novel amylase which also has an activity of endotype-hydrolyzing one or more xcex1-1,4 linkages in the molecular chain of the substrate as well as the above-described activity.
Moreover, the present invention provides a process for producing aforementioned amylase, wherein a bacterium capable of producing the above amylase of the present invention is cultivated in a culture medium, and then the amylase is isolated and purified from the culture on the basis of an activity-measuring method in which the substrate is a trehaloseoligosaccharide, and the index is the activity of producing xcex1,xcex1-trehalose.
Inventors allowed the above amylase of the present invention in combination with the aforementioned transferase of the present invention to act on a glucide raw material such as starch, starch hydrolysate, and maltooligosaccharides, and found that xcex1,xcex1-trehalose can be efficiently produced thereby with a high yield.
Accordingly, the present invention also provides a process for producing xcex1,xcex1-trehalose, wherein the above amylase and transferase of the present invention are used in combination.
Additionally, an object of the present invention is to provide a novel amylase and a gene coding for the same.
Further, another object of the present invention is to provide a recombinant novel amylase and a process for producing the same by using the aforementioned gene.
Moreover, another object of the present invention is to provide a process for producing xcex1,xcex1-trehalose by using a recombinant novel amylase.
Therefore, the gene coding for the amylase according to the present invention comprises a DNA sequence coding for a novel amylase which has the following activities:
(1) An activity of endotype-hydrolyzing an xcex1-1,4 glucoside linkage in a sugar chain;
(2) an activity of acting on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end are xcex1-1,4-linked glucose residues, so as to liberate principally monosaccharides and/or disaccharides by hydrolyzing the substrate from the reducing end side; and
(3) a principal activity of acting on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three sugar units from the reducing end side are glucose residues and the linkage between the first and second glucose residues from the reducing end side is xcex1-1,xcex1-1while the linkage between the second and third glucose residues from the reducing end side is xcex1-1,4, so as to liberate xcex1,xcex1-trehalose by hydrolyzing the xcex1-1,4 linkage between the second and third glucose residues.
Further, the recombinant novel amylase according to the present invention is a product achieved by expression of the above-described gene.
Furthermore, the process for producing xcex1,xcex1-trehalose according to the present invention comprises a step to put the above-described recombinant novel amylase and a novel transferase into contact with a saccharide of which at least three glucose residues from the reducing end are xcex1-1,4-linked, wherein the transferase can act on a substrate saccharide, the substrate saccharide being composed of at least three sugar units wherein at least three glucose residues from the reducing end are xcex1-1,4-linked, so as to transfer the first xcex1-1,4-linkage from the reducing end into an xcex1-1,xcex1-1 linkage.